The proposed research concerns N-methyl-D-aspartate receptors (NMDARs), brain proteins that are activated by the neurotransmitter glutamate and mediate communication between neurons. NMDARs are expressed by nearly every neuron in mammalian brains, and are required for normal brain function. NMDARs also are involved in many human disorders including Alzheimer's disease, schizophrenia, and cell death following stroke. There are several important drugs in common clinical use that act by binding to NMDARs, and there is optimism that new, more effective NMDAR-targeted drugs can be developed. Most clinically useful drugs that bind to NMDARs act as channel blockers, compounds that block current flow though the ion channel formed by NMDARs. The mechanisms by which channel blockers interact with NMDARs are not fully understood. In the proposed research we will examine a previously unknown path by which NMDAR channel blockers can access the channel: by entering the plasma membrane, and then transiting from the membrane into the ion channel. We will combine multiple approaches to uncover the characteristics and implications of channel blocker transit from membrane to channel. We will use electrophysiological approaches to record NMDAR activity from cells modified to express specific NMDAR subtypes, and will examine the properties of several channel blockers used clinically or as research tools. Similar approaches will be used to study channel block of native NMDARs in cultured neurons. We will use computational techniques in two ways: to model the mechanism by which blockers associate with NMDAR channels, and to create models of NMDAR structure to predict the likely path taken by channel blockers as they transit from membrane to channel. We then will test predictions of our structural models using molecular biological techniques to change the chemical makeup of NMDARs, followed by electrophysiological recording of resulting changes in channel blocker actions. Finally, we will use newly synthesized channel blocking compounds to help critically test our hypotheses, and to explore how the structure of channel blockers affects their interaction with NMDARs. The implications of the proposed research will be broad. We will reveal the basic properties of a newly discovered pharmacological mechanism by which an important class of inhibitors can interact with NMDARs. Understanding of this new mechanism of NMDAR inhibition is likely to provide insights into how other types of drugs interact with NMDARs, and into mechanisms that underlie modulation of voltage-gated channels. We will develop structural models of NMDARs of broad utility. We will use our structural models to predict the path by which drugs transit from membrane to channel and test our predictions experimentally. We will provide new information about the inhibitory mechanisms of both well-known and newly synthesized NMDAR channel blockers. We believe the knowledge gained from the proposed research will enable future design and synthesis of improved drugs to treat the many disorders in which NMDARs are implicated.